A competitive PCR assay involving the use of bronchoalveolar lavage (BAL) samples for the diagnosis of invasive pulmonary aspergillosis (IPA) was developed. For this purpose, a 1-kb mitochondrial DNA fragment of Aspergillus fumigatus was sequenced. The primers used allowed amplification of A. fumigatus, A. flavus, A. terreus, and A. niger DNAs but not DNAs of other fungi and yeasts. BAL samples from 55 consecutively enrolled patients were tested. Three samples were excluded because of failure of correct amplification of the internal competitive control. Of 28 immunocompromised patients, 6 were PCR positive; 3 died of IPA and their BAL cultures yielded A. fumigatus; and 3 were culture negative and did not develop IPA. Of 15 human immunodeficiency virus-positive patients and 9 immunocompetent patients, 5 and 4, respectively, were both PCR positive and culture negative, and none developed aspergillosis. Thus, PCR confirmed IPA in three patients but gave positive results for 25% (12 of 49) of the patients who did not develop aspergillosis. The predictive value of PCR-positive results seems low for patients at risk for aspergillosis. Moreover, the risk of contamination of reaction buffers or biological samples with Aspergillus conidia seems high and has to be weighed in regard to the potential diagnostic benefit of PCR testing as a routine procedure.

The prevalence of pulmonary toxoplasmosis was assessed by a prospective analysis of 144 bronchoalveolar lavage (BAL) samples using competitive polymerase chain reaction (PCR) to avoid false-negative results due to PCR techniques. Six samples were excluded because they contained amplification reaction inhibitors. None of the samples from the 37 immunocompetent patients and only 1 sample (1.7%) from the 59 immunocompromised patients without human immunodeficiency virus infection were PCR-positive. In contrast, Toxoplasma gondii DNA was found in 6 (14%) of 42 samples from patients with AIDS. All 6 patients had < or = 40 CD4 cells/microL and anti-Toxoplasma antibodies, and 5 had other sites of Toxoplasma infection. Six other AIDS patients who had received treatment for cerebral toxoplasmosis were PCR-negative. Thus, pulmonary toxoplasmosis is frequent in AIDS patients who have other sites of Toxoplasma infection and low CD4 lymphocyte counts and who are not receiving prophylaxis.

Candida glabrata is one of the most important causes of nosocomial fungal infection. We investigated, using a multiplex PCR, three polymorphic microsatellite markers, RPM2, MTI, and ERG3, in order to obtain a rapid genotyping method for C. glabrata. One set of primers was designed for each locus, and one primer of each set was dye labeled to read PCR signals using an automatic sequencer. Eight reference strains including other Candida species and 138 independent C. glabrata clinical isolates were tested. The clinical isolates were collected from different anatomical sites of adult patients either hospitalized in different wards of two different hospitals or not hospitalized. Since C. glabrata is haploid, one single PCR product for each PCR set was obtained and assigned to an allele. The numbers of different alleles were 5, 7, and 15 for the RPM2, MTI, and ERG3 loci, respectively. The number of allelic associations was 21, leading to a discriminatory power of 0.84. The markers were stable after 25 subcultures, and the amplifications were specific for C. glabrata. A factorial correspondence analysis did not indicate any correlation between the 21 multilocus genotypes and the clinical data (source, sex, ward, anatomical sites). Microsatellite marker analysis is a rapid and reliable technique to investigate clinical issues concerning C. glabrata. However, its discriminatory power should be improved by testing other polymorphic microsatellite loci.Epidemiological surveillance of candidemia shows that although Candida albicans remains the main species, Candida glabrata is the second leading species recovered from blood cultures in Europe and in the United States (20, 28), especially in intensive care units (9). The same phenomenon is observed for recurrent vaginitis (25). Concern arises about this increase in the incidence of C. glabrata infections because of its frequent decreased sensitivity to azoles compared to C. albicans (14,22) and the high mortality rate of bloodstream infections (28,29).Investigations for C. glabrata have not been as extensive as for C. albicans. The epidemiology is thought to be similar to C. albicans (10). However, few tools are available to study the source of contamination, cross-contamination of patients at risk, and the biology of this yeast, which has, unlike C. albicans, a haploid genome (10). Studies of population structure have begun to emerge using enzymatic and randomly amplified polymorphic DNA typing (6) or sequencing of the cytochrome c oxidase subunit 2 gene (24). Recently, a technique based on the sequencing of six variable-locus genes, or multilocus sequence typing (MLST), has been proposed as a powerful typing method (7). Microsatellites represent another class of genetic markers, defined as short tandem repeats of two to six nucleotides, known to be highly polymorphic (30). Primarily developed for human genetics, they are used for fungi (15) and more specifically for pathogenic fungi such as C. albicans (3-5, 19, 23, 26) and Aspergillus fumigatus (1, 2, 16, 17).We descri...

Toxoplasma reactivation is a life-threatening complication of allogeneic stem cell transplantation. A poor prognosis is probably linked to a difficult diagnosis, based on the detection of evidence of parasites in tissue. We developed a real-time PCR test using fluorescence resonance energy transfer hybridization probes to detect and quantify Toxoplasma gondii DNA in serum. This PCR test gave reproducible quantitative results over a dynamic range of from 0.75 × 106 to 0.75 parasites per PCR mixture. Serial samples from four patients with toxoplasma reactivation were evaluated. Three patients had several consecutive PCR-positive samples which corresponded to ≤0.75 parasites. These three patients became PCR negative during trimethoprim-sulfamethoxazole therapy but never developed clinically apparent toxoplasmosis. In contrast, one patient had an increasing PCR signal, from 1 to 396 parasites in 12 days, and developed cerebral symptoms. The parasite count decreased to 5 parasites in 3 days after pyrimethamine-clindamycin treatment. Real-time quantitative PCR is useful for diagnosis and follow-up of toxoplasma reactivation.